The proposed research aims firstly to isolate and purify each of multiple enzymes with peptide hydrolase activity from the brush border and cytosol of human and rat intestinal epithelial cells by the use of various chromatographic methods. When purification of the enzymes is achieved, a detailed analysis of kinetic properties of the enzymes and of factors which affect enzyme activity will be performed to elucidate the mechanisms by which these enzymes are regulated. A second aim of the proposed research is to determine the spatial relationship between the brush border peptide hydrolases and the brush border membranes by the use of physicochemical study of enzyme molecules and the method of lactoperoxidase catalyzed iodination of the surface proteins of the intact cells compared to those of the isolated surface membranes. Furthermore, the kinetics of peptide transport and their relationship to peptide hydrolysis by the brush border enzymes will be studied using isolated vesicular membranes in order to elucidate the functional significance of the brush border peptide hydrolases. BIBLIOGRAPHIC REFERENCES: Kim, Y.S. & E.J. Brophy. Rat intestinal brush border membrane peptidases. I. Solubilization, purification and physico-chemical properties of two different forms of the enzyme. J. Biol. Chem. In press, 1976. Kim, Y.S., E.J. Brophy & J.A. Nicholson. Rat intestinal brush border membrane peptidases. II. Enzymatic properties, immunochemistry and interactions with lectins of two different forms of the enzyme. J. Biol. Chem. In press, 1976.